Project Summary Abnormal mRNA metabolism (e.g., mRNA translation and decay) is detrimental to cellular function and has emerged as central to the molecular pathogenesis of many human diseases, such as cancer and neurological diseases. These diseases impose a devastating burden on individuals, as well as their families and communities. Unfortunately, treatment options for many remain limited. Thus, it is urgent to understand how mRNA metabolism is regulated under physiological conditions and how its dysregulation causes diseases. Over the past 5 years, studies on mRNA metabolism have revealed critical functions of various chemical modifications in mRNAs, including pseudouridine (?), 5-methylcytosine (m5C), and N6-methyladenosine (m6A), which has led to the emerging field of epitranscriptomics. m6A is the most abundant internal mRNA modification and is present in over 25% of human mRNAs. Genetic knockout studies of the m6A catalyzing enzymes METTL3 and METTL14 have revealed crucial functions of m6A in the regulation of mRNA metabolism in development and neuronal function. The m6A is co-transcriptionally deposited and highly enriched in 3'-untranslated regions (UTRs) and near stop codons. It has been demonstrated that both the level and location of m6A sites on transcripts are critical for defining the functions m6A in mRNA metabolism. However, fundamental questions remain unanswered: how cells control the level and location of m6A deposition in mRNAs and how dysregulation of this process contributes to disease pathogenesis. Our preliminary studies demonstrate that arginine methylation of METTL14 plays a critical role in maintaining the homeostasis of m6A deposition in cells and that an X-linked mental retardation (XLMR)-associated demethylase is involved in the regulation of METTL14 methylation. Based on these findings we hypothesize that the dynamic regulation of METTL14 arginine methylation by RNAPII through the recruitment of a methyltransferase and demethylase plays a critical role in controlling the level and location of m6A deposition and that dysregulation of this pathway underlies the molecular pathogenesis of impaired neuronal function in XLMR. We will 1) define the role of METTL14 arginine methylation in co-transcriptional m6A deposition; 2) determine the molecular mechanism by which METTL14 arginine methylation is regulated; and 3) investigate the impact of dysregulation of METTL14 arginine methylation on neuronal function. The results from our proposed studies will provide conceptual advances by revealing a novel molecular pathway for transcription- coupled regulation of epitranscriptomics. Identifying regulators of this process will have significant impacts on basic, translational, and clinical research of human diseases, in which loss of function of these regulators have been identified.